DNA shuffling of Cry proteins

M. Madan Babu and Dr. M. Geetha, Center for Biotechnology, Anna University

Introduction:

Bacillus thuringiensis (Bt), is a gram-positive soil bacterium, with a genome size of 2.4 to 5.7 million basepairs. The prevalence of this strain is not restricted and has been isolated worldwide from many habitats, including soil, stored-product dusts, insects, deciduous and coniferous leaves. Bacillus thuringiensis forms parasporal crystals during the stationary phase of its growth cycle. These crystals are specifically toxic to certain orders and species of insects, like Lepidoptera, Diptera, and Coleoptera. Many different strains of Bt have been shown to produce these inclusions of insecticidal crystal protein (ICP). Bt also produces antibiotic compounds that have antifungal activity. (1)

During sporulation, it synthesizes a cytoplasmic inclusion containing one or more proteins that are toxic to insect larvae. Upon completion of sporulation the parent bacterium lyses to release the spore and the inclusion. In these inclusions, the toxins exist as inactive protoxins. When the inclusions are ingested by insect larvae, the alkaline pH solubilizes the crystal. The protoxin is then converted in to an active toxin after processing by the host proteases present in the midgut. (1)

It has been indicated that the activated toxin binds to insect-specific receptors exposed on the surface of the plasma membrane of midgut epithelial cells and then inserts into the membrane to create transmembrane pores that cause cell swelling and lysis and eventually death of the insect. (1)

Due to their high specificity for these unique receptors on the membrane of the gut epithelial cells, these toxins (delta-endotoxins) are harmless to non-target insects and the end-user and are compatible with integrated pest management programs. The fact that they are proteins ensures that they are readily biodegraded.

The Cry Gene Family:

These toxins can be categorized under the d-endotoxins, which is highly specific to only certain insects. The family of genes coding for this toxin is the Cry gene family. A common characteristic of the cry genes is their expression during the stationary phase. Their products generally accumulate in the mother cell compartment to form a crystal inclusion that can account for 20-30% of the dry weight of the sporulated cells. The high level of synthesis and coordination with the stationary phase are controlled by a variety of mechanisms occurring at the transcriptional, posttranscriptional and posttranslational levels. (1)

In recent years, various Cry gene products have been discovered, and a phylogenetic tree was generated. The figure below gives an idea about the relatedness of the Cry genes (2). (revised nomenclature for the Cry genes was used.)

Fig 1. Relatedness Dendrogram of Bt crystal proteins on the basis of alignments of the full-length protein sequences. More closely related sequences or groups are connected by branch points closer to the right. Each protein is described by a name and the corresponding GenBank accession number of its DNA sequence.

Bacillus thuringiensis toxins: regulation, activities and structural diversity. H. Ernest Schnepf. Current Opinion in Biotechnology, 1995, 6:305-312.

It was clearly observed that there was a high degree of sequence similarity among the proteins in the N-terminal regions which, confers toxicity to the protein. As an illustration, the aminoacid sequences of Cry proteins from different subspecies of the B.t strain was compared and a sequence alignment was done using the software CLUSTALW. It was observed that not only the N-terminal region was conserved, but there were blocks in the C-terminal region, which were also highly conserved.

Fig 2. Highly conserved regions of the aminoacid sequence for the cry proteins among different subspecies. (The genes are Cry1B, Cry1A, Cry1D and Cry4B in order, all proteins weigh approximately 130 kd and are composed of approximately 1170 Aminoacids).

For these conserved blocks, the consensus sequence denotes the position at which atleast 75% of the aligned proteins in the group have an identical or conserved aminoacid (indicated by shading). An uppercase letter within the consensus sequence indicates that atleast 75% of the residue at that position are identical, while a lower case letter indicates that atleast 75% of the residue are conserved. Conserved aminoacids are those that fall into the following groups:

a: A, G, S, T or P :: d: D, E, N or Q :: f: F, W or Y :: i: I, L, M or V and k: K or R.

 

Structure-Function Interpretation of the Cry Proteins:

The Cry toxin has three domains which are, from N to C terminus, a seven helix bundle, (Domain I), a triple anti-parallel beta sheet domain (Domain II) and a beta-sheet sandwich (Domain III). (1)

The core of the molecule is built from five sequence blocks, which are a highly conserved feature of all the Bt toxins indicating that all the proteins in this Cry family will adopt the same general fold.

The long, hydrophobic and amphipathic alpha helices of Domain I is equipped for transmembrane pore formation. The seven alpha helix domain I structure resembles the pore forming domain of Colicin A and is important for the membrane insertion step.

Pore formation is initiated by insertion of a helical hairpin (alpha4/alpha5) from domain I with subsequent association of alpha4/alpha5 hairpins from several molecules to form an oligomeric helical bundle pore with a radius of 5-10 Angstroms.

Before one or more of these Cry helices can insert into the membrane to initiate oligomerization and pore formation, a major conformational change must occur, since in the water soluble pre-insertion form all the hydrophobic faces of the Cry Domain I helical bundle face inwards.

Membrane penetration occurs in two steps: binding to a specific receptor exposed on the membrane surface, followed by insertion of the delta-endotoxin protein into the membrane leading to pore formation.

The three beta sheet structure (beta prism) of domain II is involved in receptor binding and specificity determination. This is further supported by reports that domain II shared the same structural fold with three carbohydrate binding proteins: the vitelline membrane outer layer protein I from hen's eggs, the plant lectin jacalin and the Maclura pomifera agglutinin.

Domain III of the Bt toxin (see below) may also be a determinant of insect specificity/receptor binding.

Fig 3. Schematic ribbon diagram structure of the CryA toxin. The three domains and their suggested functions are indicated. Shaded segments correspond to the five conserved sequence blocks (1–5). The position of the b-sheets of domain II are indicated near the protruding loops of each sheet

The striking similarity between the structure of domain II of the Bt toxins and the three dimensional structures of two known lectins suggests that insecticidal specificity might be determined by the carbohydrate affinity of the domain II lectin fold. A recent discovery that domain III is also a lectin-like domain suggests that the insecticidal specificity of these toxins could be determined by two lectin-like domains acting in concert or independently.

To fully realize the potential of Bacillus thuringiensis d-endotoxins as biopesticides, progress is required in several areas. First, we must increase the yield or efficiency of toxin protein production. Second, we must gain a sufficient understanding of the mechanism of toxicity to allow engineering of the toxins for maximum activity. Third, we must continue to isolate new strains with novel toxin structures and activities either on known B. thuringiensis targets or on pests thought to be insensitive to B. thuringiensis. And fourth, we must gain a better understanding of the mechanism, and management, of insect resistance to B. thuringiensis toxins. All the requirement can be easily met by the process of DNA shuffling of the Cry genes.

 

DNA shuffling:

DNA shuffling is a powerful process for directed evolution, which generates diversity by recombination. DNA family shuffling mimics and extends classical breeding methods by recombining more than two parental genes, or genes from different species, in a single DNA shuffling reaction. In this method, the gene is subjected to random mutations and is then screened for improved ones.

The previous methods of DNA shuffling (3) incorporated, the Single Sequence Shuffling, where a pool of homologous genes with different point mutations (induced by error prone PCR, or by Oligo nucleotide directed mutagenesis) is cut in to fragments by DNase I Digestion, followed by reassembly of the fragments in to full genes by using PCR with and without primers (discussed below).

It was found that the sequence space for searching for this method was very less when compared to the sequence space available in DNA family shuffling. (4)

Fig 4: Searching sequence space by family shuffling versus by single sequence shuffling. Single sequence shuffling yields clones with a few point mutations and the library members are 97-99% identical. Family shuffling causes sequence block exchange, which yields genes that have greater diversity. At equal library size, the sampling space is larger for DNA family shuffling than the single sequence shuffling, thereby allowing promising areas to be found.

DNA shuffling of a family of genes from diverse species accelerates directed evolution. Willem P.C. Stemmer etal. Nature 1998,391:288-291.

The process of DNA family shuffling (4,5) is illustrated in the diagram (see next page). A detailed protocol of how this is carried out is discussed in the experimental protocol section.

 

Fig 5. DNA shuffling methodology. The first step of this method is to randomly fragment a population of related genes using DNAse I. This produces fragments of various lengths that, after denaturation, hybridize to form an equal mixture of 5' and 3' overhangs. Using PCR techniques, the 5' overhang fragments can be extended by Taq DNA polymerase—leaving the 3' overhang fragments unaffected. As a consequence of this extension, the average fragment length increases during each cycle. Recombination occurs, when a fragment derived from one template primes a template with a different sequence. Green dots represent beneficial mutations and red dots represent deleterious mutations. The colored bars indicate recombinations of portions of three parents into recombinant progeny.

Applications of DNA shuffling to pharmaceuticals and vaccines. Phillip A Patten, Russell J Howard, Willem PC Stemmer. Current Opinion in Biotechnology 1997, 8:724-733.

Objective:

The following characteristics of the Bt toxin are to be achieved, using DNA family shuffling.

Experimental Protocol:

The following is the detailed experimental protocol adopted for DNA Shuffling of the Cry genes.

1. Get DNA sequences of Cry proteins of interest and design degenerate primers : Download the sequences of the Cry protein of interest from the sequence database (from EMBL and SWISS-PROT) and do a sequence alignment of them using CLUSTALW, to find out conserved regions in the sequences and design the primer.

2. Use these primers to amplify the genes in the chosen Bt subspecies : Use the designed primer and perform a PCR to amplify the genes present in the Bt subspecies. Run an agarose gel electrophoresis and elute the amplified genes in the gel. Purification of the fragments is done, by transferring on to DE81 ion-exchange paper (Whatman), elution with NaCl, followed by ethanol precipitation.

3. Clone it in E.coli and express it : Clone the genes in E.coli, in a suitable cloning site and express the protein to check its activity. Screen out the ones, which do not produce functional protein and retained the properly cloned colonies. (Screening is done, based on the protein’s ability to bring about toxicity).

4. Amplify the genes in these colonies using the previously designed primer in a PCR.

5. Substrate for the DNA shuffling experiment : The substrate for the experiment will be the PCR amplified genes in which the primers are removed, to get only the full genes.

6. DNase I digestion of the substrate and purification of bases, 10-50 bases in length : Using DNase I, digest the substrate in to small fragments. Purify the fragments by elution (whose, length are 10-50 bases in length) after running an agarose gel electrophoresis. The purification is done, by transferring on to DE81 ion-exchange paper (Whatman), elution with NaCl, followed by ethanol precipitation.

7. PCR without primers : Resuspend the purified fragments in a PCR mixture without primers, and perform the reaction. The templates are the sequences purified in step 4. Each cycle causes a template switch and provides a ground for recombination of fully functional blocks. This results in the sequences to be almost unique.

8. PCR with primers : In order to selectively amplify the full genes, which have been obtained, perform a PCR with the originally designed primers.

9. Purification of full genes :Run an agarose gel electrophoresis, to get a sharp band, corresponding to the full gene. Purify it by transferring onto DE81 ion-exchange paper (Whatman), followed by elution with NaCl, and precipitation using ethanol.

10. Cloning : After digestion of the PCR products with suitable restriction enzymes and gel purification, clone them in to E.coli cells.

11. Design a high throughput screen to select functional proteins (qualitatively).

12. Select best mutants by quantitative analysis.

 

References:

1. Schnepf H.E, etal. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews. 1998, 62:775-806.
2. Schnepf H.E, etal. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiology and Molecular Biology Reviews.1998, 62:807-813.
3. Stemmer W.P.C. DNA Shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. 1994, 91:10747-10751.
4. Stemmer W.P.C. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature. 1998, 391:288-291.
5. Patten P.A, Howard R.J and Stemmer W.P.C. Applications of DNA shuffling to pharmaceuticals and vaccines. Curr. Opin. Biotechnol.1997, 8:724-733.
6. Schnepf H.E, etal. Bacillus thuringiensis toxins: regulation, activities and structural diversity. Curr. Opin. Biotechnol 1995, 6:305-312.
7. Stemmer W.P.C, etal. Evolution of a cytokine using DNA family shuffling. Nature. 1999, 17: 793-797.
8. Stemmer W.P.C. Rapid evolution of a protein In vitro by DNA shuffling. Nature. 1994, 370:389-391.
9. Arnold F.H. and Zhao H. Functional and nonfunctional mutations distinguished by random recombination of homologous genes. Proc. Natl. Acad. Sci. 1997, 94:7997-8000.
10. Lacy D.B and Stevens R.C. Unraveling the structures and modes of action of bacterial toxins. Curr. Opin. Struct. Biol.1998, 8:778-784.
11. Stemmer W.P.C. DNA shuffling of subgenomic sequences of subtilisin. Nature Biotech. 1999, 17:893-896.
12. Ellington A.D, etal. Directed evolution of the surface chemistry of the reporter enzyme b-glucuronidase. Nature Biotech. 1999, 17:696-701.